Guanidinium Toxins as Molecular Probes for NaV Study

胍毒素作为 NaV 研究的分子探针

• 批准号: 9330901
• 负责人: Justin Du Bois
• 金额: $ 41.15万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9330901
• 关键词: Acute Pain; Affect; Affinity; Analgesics; Binding; Binding Sites; Biochemical; Biochemistry; Biological Phenomena; Cell membrane; Cells; Chemicals; Collection; Complex; Cysteine; Data; Development; Docking; Electricity; Electrophysiology (science); Engineering; Event; Experimental Designs; Fluorescent Probes; Functional disorder; Genetic Engineering; Goals; Homology Modeling; Human Pathology; Imaging technology; Individual; Investigation; Investigational Drugs; Ion Channel; Ions; Kinetics; Knowledge; Label; Lead; Ligands; Location; Maintenance; Measures; Membrane; Methods; Microscopy; Modeling; Molecular; Molecular Probes; Movement; Mutagenesis; Natural Products; Nature; Nerve; Nerve Block; Nervous system structure; Neurons; Oral cavity; Organism; Output; Pain management; Pathway interactions; Pharmaceutical Preparations; Poison; Post-Translational Protein Processing; Process; Prokaryotic Cells; Protein Engineering; Protein Isoforms; Proteins; Reagent; Recombinants; Reporting; Research Design; Resolution; Roentgen Rays; Role; Saxitoxin; Shapes; Signal Transduction; Site; Sodium Channel; Source; Spatial Distribution; Structure; Structure-Activity Relationship; Takifugu; Technology; Tetrodotoxin; Time; Toxin; Vestibule; Work; analog; base; cell injury; chemical synthesis; chronic pain; design; experimental study; extracellular; fluorescence imaging; gonyautoxins; guanidinium; inhibitor/antagonist; injured; insight; interest; mutant; nanomolar; nerve injury; neurotransmission; new therapeutic target; next generation; novel therapeutics; operation; painful neuropathy; protein complex; protein degradation; receptor; response; small molecule; three dimensional structure; tool; trafficking; voltage; zetekitoxin AB

PROJECT SUMMARY Proper neuronal function relies on the tightly regulated expression and discrete localization of voltage-gated sodium ion channels (NaVs), large protein complexes that control the movement of ions across cell membranes. A desire to better understand the role of NaVs in electrical signal conduction and the relationship between channel disregulation and specific human pathologies motivates the development of high precision reagents for their study in living systems. Real-time investigations of NaVs in live neuronal cells, however, are limited by the lack of available methods with which to modulate the function of individual NaV subtypes and to mark their cellular distributions and membrane expression levels. We are developing small molecule probes for NaV studies based on naturally occurring guanidinium toxins – saxitoxin, gonyautoxins, and zetekitoxin. These agents function as molecular `corks' to occlude the extracellular mouth of the ion conductance pore. De novo chemical synthesis makes available modified forms of these toxins, which we will use in combination with protein mutagenesis and electrophysiology to gain insights into the three- dimensional structure of the toxin binding site. Such information is needed to advance a high fidelity NaV homology model, and will empower the rational design of toxin derivatives that display selective inhibition of individual NaV isoforms. Our structural investigations of toxin binding are informing the development of new fluorescent imaging and affinity-based tools, which will be utilized to explore dynamic events associated with NaV function. We wish to understand how modulation of NaV membrane expression and post-translational protein modifications influence the input-output responsiveness of neuronal cells following nerve injury. Toxin-derived fluorescent probes will be prepared and used to measure the spatial distributions and concentrations of membrane-inserted NaVs in live cells. These investigations will provide a quantitative analysis of how NaV structure (i.e., post- translational modification), ion gating, membrane distribution, and protein turnover rates are altered in neuronal cell injury models. In addition, our experimental design will allow us to assess the influence of investigational drugs, protein factors, and/or other small molecules on regulating NaV trafficking and restoring proper neuronal signaling. Ultimately, this work could lead to the identification of new therapeutic targets or lead compounds for pain treatment.

项目概要 适当的神经元功能依赖于严格调节的表达和离散定位 电压门控钠离子通道 (NaV)，控制运动的大型蛋白质复合物 离子穿过细胞膜。渴望更好地了解 NaV 在电气中的作用 信号传导以及通道失调与特定人体的关系 病理学推动了高精度试剂的开发，用于活体研究 系统。然而，对活神经元细胞中 NaV 的实时研究受到以下因素的限制： 缺乏可用的方法来调节单个 NaV 亚型的功能并 标记它们的细胞分布和膜表达水平。 我们正在开发基于自然发生的 NaV 研究的小分子探针 胍毒素——石房蛤毒素、膝沟藻毒素和泽特毒素。这些代理的作用是 分子“软木塞”堵塞离子电导孔的细胞外口。从头 化学合成提供了这些毒素的修饰形式，我们将在 结合蛋白质诱变和电生理学，深入了解这三者 毒素结合位点的空间结构。需要此类信息来推进高水平 保真NaV同源模型，将赋能毒素衍生物的合理设计 显示出对单个 NaV 同种型的选择性抑制。 我们对毒素结合的结构研究为新荧光的开发提供了信息 成像和基于亲和力的工具，将用于探索相关的动态事件 具有NaV功能。我们希望了解 NaV 膜表达的调节和 翻译后蛋白质修饰影响神经元的输入输出反应 神经损伤后的细胞。毒素衍生的荧光探针将被制备并用于 测量活细胞中插入膜的 NaV 的空间分布和浓度。 这些研究将对 NaV 结构（即后 翻译修饰）、离子门控、膜分布和蛋白质周转率 神经元细胞损伤模型中的改变。此外，我们的实验设计将使我们能够 评估研究药物、蛋白质因子和/或其他小分子对 调节 NaV 运输并恢复适当的神经元信号传导。最终，这项工作可以 导致识别新的治疗靶点或用于疼痛治疗的先导化合物。